The ventral tegmental area (VTA) is thought to play an important role in reward function. Two populations of neurons, containing either dopamine (DA) or gamma-amino butyric acid (GABA), have been extensively characterized in this area. However, recent electrophysiological studies are consistent with the notion that neurons that utilize neurotransmitters other than DA or GABA are likely to be present in the VTA. Given the pronounced phenotypic diversity of neurons in this region, we have proposed that additional cell types, such as those that express the neurotransmitter glutamate may also be present in this area. Thus, by using in situ hybridization histochemistry we investigated whether transcripts encoded by genes for the two vesicular glutamate transporters, VGluT1 or VGluT2, were expressed in the VTA. We found that VGluT2 mRNA but not VGluT1 mRNA is expressed in the VTA. Neurons expressing VGluT2 mRNA were differentially distributed throughout the rostro-caudal and medio-lateral aspects of the VTA, with the highest concentration detected in rostro-medial areas. Phenotypic characterization with double in situ hybridization of these neurons indicated that they rarely coexpressed mRNAs for tyrosine hydroxylase (TH, marker for DAergic neurons) or glutamic acid decarboxylase (GAD, marker for GABAergic neurons). Based on results described here, we concluded that the VTA contains glutamatergic neurons that in their vast majority are clearly non-DAergic and non-GABAergic.[unreadable] To investigate whether VTA glutamatergic neurons establish local synapses, we marked axon terminals of VTA neurons by local injection of the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHAL) and used double immunolabeling and electron microcopic analysis to determine if PHAL-positive terminals within the VTA contained VGluT2. PHAL-positive terminals established either asymmetric or symmetric synapses, predominantly on dendrites, more than half of all PHAL-positive terminals that made recognizable synapses contained VGluT2. Double labeled PHAL/VGluT2 terminals established predominantly asymmetric synapses while most PHAL-positive terminals lacking VGluT2 formed symmetric synapses. To determine whether DAergic neurons receive glutamatergic inputs from local neurons, we used triple fluorescence immunolabeling with antibodies against PHAL, VGluT2 and tyrosine hydroxylase (TH, a marker for DAergic neurons). By confocal fluorescence microscopy we observed double labeled PHAL/ VGluT2 varicosities contacting TH-positive dendrites. By electron microscopy, we further determined that double labeled PHAL/VGluT2 axon terminals formed synaptic contacts on dendrites of both TH-immunoreactive and TH-negative cells. In whole cell recordings of VTA neurons we observed that blocking local action potential activity significantly decreased the frequency of synaptic glutamatergic events in DAergic and non-DAergic neurons. In summary, we provide anatomical and electrophysiological evidence that local glutamatergic neurons establish intrinsic synapses within the VTA.